Aim: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

Methods: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.

fig6: WSS25 inhibits the RANKL- or BMP-2-induced phosphorylation of Smad1/5/8 in RAW264.7 cells. (A, B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL) for 22 h. Cells were then lysed. The phosphorylation Smad1/5/8 was detected by Western blot. (C) RAW264.7 cells were incubated with BMP-2 (100 ng/mL) for the indicated times. The cells were lysed and the phosphorylation of Smad1/5/8 was detected by Western blot. (D) RAW264.7 cells were stimulated with BMP-2 (100 ng/mL) and treated with WSS25 (10 μg/mL) or noggin (250 ng/mL) for 60 min. Then, the cells were lysed and the extracts were probed with anti-pSmad1/5/8 antibody. (E) RAW264.7 cells were incubated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on the indicated days. BMP-2 expression was detected by immunoblotting.

Mentions:
As we have previously reported, WSS25 binds to BMP-2 to block downstream Smad1/5/8 and Id1 signaling pathways26. To explore the mechanisms contributing to the inhibitory effect of WSS25 on osteoclast formation, RAW264.7 cells were stimulated with RANKL and treated with WSS25 for 22 h, followed by measurements of the phosphorylation of Smad1/5/8 by Western blot analysis. The phosphorylation of Smad1/5/8 was significantly promoted by RANKL. However, 10 μg/mL of WSS25 efficiently blocked RANKL-induced phosphorylation of Smad1/5/8 (Figure 6A). Noggin, a BMP-2 endogenous antagonist, had an inhibitory effect similar to that of WSS25 (Figure 6B). As the direct intracellular effectors of BMP-2, phosphorylated Smad1/5/8 was also promoted by exogenous BMP-2 in RAW264.7 cells (Figure 6C). However, WSS25 or noggin blocked the BMP-2 induced phosphorylation of Smad1/5/8 (Figure 6D).

fig6: WSS25 inhibits the RANKL- or BMP-2-induced phosphorylation of Smad1/5/8 in RAW264.7 cells. (A, B) RAW264.7 cells (1×106 cells/mL) were incubated with RANKL (50 ng/mL) in the presence or absence of WSS25 (10 μg/mL) or noggin (250 ng/mL) for 22 h. Cells were then lysed. The phosphorylation Smad1/5/8 was detected by Western blot. (C) RAW264.7 cells were incubated with BMP-2 (100 ng/mL) for the indicated times. The cells were lysed and the phosphorylation of Smad1/5/8 was detected by Western blot. (D) RAW264.7 cells were stimulated with BMP-2 (100 ng/mL) and treated with WSS25 (10 μg/mL) or noggin (250 ng/mL) for 60 min. Then, the cells were lysed and the extracts were probed with anti-pSmad1/5/8 antibody. (E) RAW264.7 cells were incubated with RANKL (50 ng/mL), in the presence or absence of WSS25 (10 μg/mL), on the indicated days. BMP-2 expression was detected by immunoblotting.

Mentions:
As we have previously reported, WSS25 binds to BMP-2 to block downstream Smad1/5/8 and Id1 signaling pathways26. To explore the mechanisms contributing to the inhibitory effect of WSS25 on osteoclast formation, RAW264.7 cells were stimulated with RANKL and treated with WSS25 for 22 h, followed by measurements of the phosphorylation of Smad1/5/8 by Western blot analysis. The phosphorylation of Smad1/5/8 was significantly promoted by RANKL. However, 10 μg/mL of WSS25 efficiently blocked RANKL-induced phosphorylation of Smad1/5/8 (Figure 6A). Noggin, a BMP-2 endogenous antagonist, had an inhibitory effect similar to that of WSS25 (Figure 6B). As the direct intracellular effectors of BMP-2, phosphorylated Smad1/5/8 was also promoted by exogenous BMP-2 in RAW264.7 cells (Figure 6C). However, WSS25 or noggin blocked the BMP-2 induced phosphorylation of Smad1/5/8 (Figure 6D).

Aim: WSS25 is a sulfated polysaccharide extracted from the rhizome of Gastrodia elata BI, which has been found to bind to bone morphogenetic protein 2 (BMP-2) in hepatocellular cancer cells. Since BMP-2 may regulate both osteoclasts and osteoblasts, here we investigated the effects of WSS25 on osteoclastogenesis in vitro and bone loss in ovariectomized mice.

Methods: RAW264.7 cells or mouse bone marrow macrophages (BMMs) were treated with RANKL to induce osteoclastogenesis, which was assessed using TRAP staining, actin ring formation and pit formation assays, as well as bone resorption assay. Cell viability was detected with MTT assay. The mRNA levels of osteoclastogenesis-related genetic markers (TRAP, NFATc1, MMP-9 and cathepsin K) were detected using RT-PCR, while the protein levels of p-Smad1/5/8 and Id1 were measure with Western blotting. WSS25 was administered to ovariectomized mice (100 mg·kg(-1)·d(-1), po) for 3 months. After the mice were euthanized, total bone mineral density and cortical bone density were measured.